PROJECT SUMMARY: Codon usage is a specific feature of each gene and each genome and impacts the fitness of each organism. In the degeneracy of the genetic code, proteins can be coded in multiple ways using different sets of synonymous codons, which are not translated equally in speed or quality. Each codon choice between the synonyms makes a demand for the supply of the tRNA with the matching anticodon. The quality of a codon-anticodon pairing interaction is determined not only by the level of the tRNA for the codon, but also by the epigenetic modifications to the tRNA that are synthesized post-transcriptionally. While most studies have focused on the abundance of tRNA as a determinant of codon usage and cell fitness, less is known about post-transcriptional modifications. In the past 5 years, my lab has focused on the N1-methylation of the guanine at position 37 that synthesizes m1G37 in tRNAs, which is required for reading-frame maintenance during protein synthesis. Loss of m1G37-tRNAs leads to accumulation of ribosomal +1-shifts, resulting in pre-mature termination of protein synthesis and ultimately cell death. A key finding of our work is that, while m1G37 is required for translation of all four codons for proline (Pro), it is essential for translation of CC[C/U] codons. Because Pro is a unique amino acid in protein synthesis, this finding offers interesting and important new biology, in which m1G37- tRNAs provide a global mechanism to control the expression of CC[C/U]-enriched genes. In the next frontier of research, we will focus on the m1G37-dependent differential translation of CC[CU] as a model to elucidate the principles by which the supply-to-demand ratio of tRNAs governs cell fitness. We will start by analysis of the balanced growth of E. coli as a reporter for genome-wide protein synthesis. We will test the predictive power of the elucidated principles in determining the human proteome. We will also address the role of m1G methylation, when placed at position 9 of a pathogenic mitochondrial tRNA (mt-tRNA), in the development of the mitochondrial disorder. By exploring the unique methodologies and conceptual frameworks that we have developed, we will address these key gaps in the field and advance our understanding of codon usage in human health and disease.